Otitis media (OM) is one of the largest public health problems in young children, and is especially devastating in developing countries. The large medical costs involved in treatment lead to research initiatives in order to understand the disease and develop better treatment plans. Animal models of OM have been developed to study different disease factors known in vivo and ex vivo. Much has been discovered about the basic immune, inflammatory and genetic mechanisms of OM using murine and other animal models. However, since human responses are unique, it is important to link animal studies to human immunity and inflammation. Humanized mice have become a valuable tool with which to study the human immune system in a mouse model. Immunodeficient mice (NSGs) are irradiated at a juvenile age to destroy bone marrow immune stem cells and are then engrafted with human immune stem cells which, with time, undergo multi-lineage development resulting in a fully functional human immune system (including T-, B-, NK-, and dendritic cells, as well as monocytes/macrophages and granulocytes). These animal models will have a number of potential uses in OM studies, including studying the role of human immune response during the course of OM, evaluating the relative abundance and functions of human immunocyte versus mouse mucosal responses in OM, and evaluation of immune responses using stem cells derived from otitis-prone versus otitis-resistant patients. We will establish a humanized mouse model of OM by characterizing the middle ear inflammatory response, leukocyte infiltration, tissue hyperplasia, bacterial clearance, biofilms, and OM recovery following inoculation with nontypeable Haemophilus influenzae (NTHi) and S.pneumoniae, as compared to wild type BALB/c mice, irradiated BALB/c mice and finally NSG mice engrafted with murine immune cells. The proposed research will pave the way to utilizing humanized mice as murine models for the study of human immunity in OM.